The high affinity receptor for IgE ("E-receptor") on mast cells and basophils plays a central role in immediate hypersensitivity reactions. Reaction of receptor-bound IgE with polyvalent antigen clusters the receptors and this stimulates cellular secretion of both preformed and newly synthesized mediators of inflammation. Our studies focus on the molecular mechanisms by which aggregation of E-receptors generate these responses. The similarity of E-receptors to other receptors of the immune system (e.g. the clonotypic receptors on T and B lymphocytes), make it likely that the significance of such studies extends beyond the IgE/mast cell system. During the past year, our principal studies have been along the following lines: 1) We utilized Chinese Hamster Ovary cells permanently transfected with E-receptors to analyze the receptors' interaction with Lyn kinase. In studies reported in the prior report and which are now published, we utilized various constructs of Lyn kinase--the enzyme responsible for phosphorylating the IgE receptor as the initiating event following aggregation--to define a specific region on the kinase that would account for the interaction. During this past year we attempted to perform the complementary experiments. That is, we used transfection techniques to see if we could identify that part of the E-receptor interacting with the kinase. Because there is evidence that the cytoplasmic portions of the "beta" and "gamma" subunits may be involved, we used genetic engineering to prepare chimeric proteins consisting of the ecto- and transmembrane domains of an irrelevant receptor chain (IL-2 alpha chain) fused to the E-receptor regions of interest. Initial results indicate that we have been able to achieve variable levels of expression and that specific interaction can be observed. 2) We previously reported on initial studies to test the hypothesis that the number of sequential perturbations that the E-receptors can stimulate should be positively correlated with ligand affinity. These studies were completed and have just been published. Several observations were of interest: a) We obtained strong evidence that a weakly binding ligand, although perfectly capable of stimulating the most proximal events, was incapable of stimulating distal events regardless of the dose used. b) With ligands of intermediate affinity, the degree to which distal responses were observable was related to the size of the aggregates formed. c) The low affinity ligand could actually inhibit the responses engendered by the high affinity ligand by sequestering the initiating Lyn kinase. All of these results strongly support our fundamental proposal that the limited availability of the kinase to the receptor is a significant factor that regulates the cellular response to receptor engagement. 3) The initial analysis of the changes in the profile of genes that are expressed in activated versus resting mast cells using Serial Analysis of Gene Expression (SAGE) methodology has been completed. The analysis revealed a) genes not previously known to be active in mast cells; b) genes not previously known; c) up-regulation of genes some of which were previously known to be up-regulated in mast cells but also many whose expression was not known to be stimulated. Approximately 20 assignments were explored further to see if their presence could be verified either by identifying their messenger or their protein product or both. In each instance, the SAGE analysis was confirmed; that is, there were no false positives. It is apparent that this is a powerful method for defining the complex and dynamic genetic expression of a cell. 4) We are extending our characterization of the phosphatases that dephosphorylate activated receptors. We have developed a practical assay that allows us to measure efficiently and with reasonable accuracy large numbers of samples to evaluate their content of phosphatase(s) having the appropriate specificity. Recent results suggest that they may be enriched if not confined to the plasma membrane.